Pseudomonas aeruginosa (P. aeruginosa) is invasive and toxigenic, produces infections in patients with abnormal host defenses, and is an important nosocomial pathogen. The pneumonia caused by P. aeruginosa is very severe and results in necrotizing pneumonia. Frequently this pneumonia is exacerbated to cause severe acute lung injury in immunocompromised patients such as AIDS patients. Intratracheal instillation of P. aeruginosa is well known as a model of pneumonia and acute lung injury. Thus, pneumonia and acute lung injury after exposure to P. aeruginosa is thought to be caused by free radicals, proinflammatory cytokines, arachidonic acid metabolites, and proteases derived from infiltrated neutrophils, activated macrophages, bacteria, and their products. Recently, free radicals have been a special focus as the final causative molecules in the pathogenesis of acute lung injury caused by P. aeruginosa infection, although there is no direct evidence of free radical generation in lung injury caused by P. aeruginosa in vivo. Therefore, using electron spin resonance (ESR) and the spin-trapping technique with alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN), we investigated in vivo free radical production by rats treated with intratracheal instillation of P. aeruginosa. Twenty-four hours after instillation of LPS, an inflammatory response was confirmed by histological analysis of neutrophil infiltration by broncho-alveolar lavage. ESR spectroscopy of lipid extract from lungs infected with P. aeruginosa for 24 hours gave a spectrum consistent with that of a carbon-centered radical spin adduct of POBN (aN= 14.86 +/- 0.03 G and aH-beta= 2.48 +/- 0.09 G). The intensity of ESR spectra was significantly increased by the infection of P. aeruginosa (Relative intensity; P. aeruginosa infection 20.71 +/- 8.67 mm, Control 6.50 +/- 1.41 mm, p < 0.01). Previous investigations have tentatively assigned this radical adduct as a product of in vivo lipid peroxidation. To further investigate the mechanism of P. aeruginosa-induced free radical generation, rats were pretreated with the phagocytic toxicant GdCl3, which significantly decreased the production of radical adducts with a corresponding decrease in neutrophil infiltration as indicated by histopathological studies. It was confirmed that free radical production in this system depended on phagocytes, which could be either activated macrophages or neutrophils. In conclusion, rats treated with intratracheal instillation of P. aeruginosa generate free radicals in the lung as demonstrated by ESR analysis.